Icariin promotes osteogenic differentiation by upregulating alpha-enolase expression

Osteogenic differentiation is a crucial biological process for maintaining bone remodelling. Aerobic glycolysis is the main source of energy for osteogenic differentiation. Alpha-enolase (Eno1), a glycolytic enzyme, is a therapeutic target for numerous diseases. Icariin, a principal active component of the traditional Chinese medicine Epimedium grandiflorum, can stimulate osteogenic differentiation. Here, we aimed to determine if icariin promotes osteogenic differentiation via Eno1. Icariin (1 μM) significantly promoted osteogenic differentiation of MC3T3-E1 cells. Icariin upregulated Eno1 protein and gene expressions during osteogenic differentiation. Moreover, ENOblock, a specific inhibitor of Eno1, markedly inhibited icariin-induced osteogenic differentiation. Futhermore, western blot assay showed that Eno1 might mediate osteogenic differentiation through the BMP/Smad4 signalling pathway. Collectively, Eno1 could be a promising drug target for icariin to regulate osteogenic differentiation.


Introduction
Bone remodelling is a dynamic process resulting from both bone formation and bone resorption [1]. The orchestrated interplay between osteoblasts and osteoclasts is crucial for bone remodelling [2]. Abnormal osteogenesis jeopardises bone homeostasis by inducing an imbalance between bone formation and bone resorption, leading to disorders, such as osteoporosis and osteoarthritis [3,4].
Energy metabolism plays a crucial role in maintaining healthy tissues [5]. Skeletal metabolic energy disorders disrupt bone metabolism balance, leading to bone-related diseases [6]. Osteoblast formation is an energy-consuming process, and the energy demands are met by active energy metabolism [7]. Aerobic glycolysis is the main source of energy during osteogenic differentiation [8]. Augmented aerobic glycolysis also promotes osteoblast differentiation [9]. Several glycolytic enzymes and metabolites, such as glucose-6-phosphate isomerase 1 and pyruvate, were reported to be involved in regulating osteoblast differentiation [10,11]. Alpha-enolase (Eno1), which catalyses the conversion between 2-phosphoglyceric acid and phosphoenolpyruvic acid, is involved in many biological functions, including cellular stress, cancer metastasis and autoantigen activities [12]. The differential expression of Eno1 leads to several diseases, including cancer, Alzheimer's disease and rheumatoid arthritis [13]. However, the role of Eno1 in the regulation of bone metabolism is unclear.
Icariin is the main ingredient of Epimedium grandiflorum, which is a traditional Chinese medicine. Icariin was proved to modulate bone remodelling through promoting osteogenic differentiation and inhibit osteoclast formation [14][15][16]. BMP/Smad signalling pathway was confirmed to be a key target for icariin efficacy both in vivo and in vitro [17,18]. When the glucose metabolism disorder occurs under the condition where the bone remodelling is destructive, such as diabetes-induced osteoporosis, BMP/Smad is closely involved in the regulation of osteoblast differentiation [19]. Given that Eno1 expression markedly changes in ovariectomised and icariin-treated ovariectomised rats (unpublished data), the relationship between Eno1, BMP/Smad signalling pathway and icariin-regulated bone metabolism is of great interest.
In the present study, we confirmed the efficacy of icariin on osteogenic differentiation using Alizarin red staining, osteogenic gene detection and alkaline phosphatase (ALP) activity measurements. Eno1 gene and protein levels were determined in icariin-induced osteogenic differentiation. Further, ENOblock, an inhibitor of Eno1, was used to explore the function of Eno1 in osteogenic differentiation and icariin efficacy. Finally, the role of BMP/Smad4 signalling in Eno1-regulated osteogenic differentiation was determined.

Cell culture
The MC3T3-E1 cell line was purchased from China Procell Biotechnology Co., Ltd. Cells were cultured in α-MEM medium containing 10% foetal bovine serum (Gibco), 100 U/ml penicillin and 100 U/ml streptomycin at 37 • C in a 5% CO 2 -saturated humidified incubator. Osteogenic differentiation was induced when cells reached more than 80% confluence. Fifth passage of MC3T3-E1 cells were induced with osteogenic differentiation culture medium (HyCyte TM , EOMX-D101, China).
The experiment was divided into two steps. To determine the optimal concentration of icariin to induce osteogenic differentiation of MC3T3-E1 cells, the cells were treated with osteogenic differentiation culture medium containing four concentrations of icariin: 0(blank control group), 0.01, 0.1 and 1 μM. To determine the role of Eno1 in osteogenic differentiation and icariin efficacy, osteogenic differentiation culture medium containing 5 μM ENOblock (MedChemExpress) was used. The cells were divided into blank control, ENOblock, icariin and icariin + ENOblock groups.

Cell viability assessment
MC3T3-E1 cells in the logarithmic growth phase were seeded into a 96-well plate at a density of 10 3 cells/well. The experiment was conducted in two steps. First, to determine the effect of ICA on cell viability, cells were cultured with or without different concentrations of icariin (0.01, 0.1 and 1 μM). Second, to determine the effect of ENOblock on cell viability, cells were fed with or without different concentrations of ENOblock (0.05, 0.5 and 5 μM). Cells were cultured at 37 • C and 5% CO 2 for 24, 48 and 72 h after treatment. 100 μL CCK8 (APExBIO, USA)was added and the cells were incubated at 37 • C for an additional 2h. Absorbance was measured at 450 nm with a microplate reader.

Alizarin red S staining
MC3T3-E1 cells were seeded into 6-well plates. After reaching 80% confluence, the cells were treated with icariin and ENOblock, as described above. After culturing the treated cells for 21 days, Alizarin red S staining was performed according to the manufacturer's instructions (Solarbio, China). Alizarin red S was destained with 10% cetylpyridinium chloride for 30 min and the absorbance at 562 nm was measured to determine the calcium deposits.

ALP activity assay
MC3T3-E1 cells were seeded into 6-well plates. The cells were cultured in osteoblast differentiation induction medium with or without different concentrations of icariin for 7 days. ALP activity was measured according to the ALP detection kit instructions (Nanjing Jiancheng Biotechnology Co., Ltd.) The enzymatic activity unit was defined as 1 mg of phenol produced per mg of lytic protein at 37 • C for 15 min as a Guinness unit.

Real-time quantitative PCR
Cells were induced for 7 days and RNA was extracted using a RNA extraction kit (Aidlab Biotech, China). The cDNA was synthesised on a Veriti-96 Well Thermal Cycler (Applied Biosystems, USA) using a Pri-meScript TM RT Reagent Kit (TaKaRa, Dalian, China). Real-time quantitative PCR (qPCR) was performed using a CFX Opus 96 (Bio-Rad, USA) with TB Green® Premix Ex Taq TM (TaKaRa, Dalian, China). The qPCR conditions were as follows: denaturation at 95 • C for 30 s, 40 cycles of PCR reaction at 95 • C for 5 s and 60 • C for 30 s. The melting curves at the end of amplification were analysed. β-actin was used as a control, and the data were calculated using the comparative Ct (2 − ΔΔCT ) method and normalized against β-actin. All the primer sequences are shown in Supplementary Table S1.

Bioinformatics analysis
Genes related to postmenopausal osteoporosis and diabetes mellitus were obtained from the GeneCards, Online Mendelian Inheritance in Man, PharmGKB, Drugbank and TTD databases. The common target genes were imported into the String database to construct a proteinprotein interaction network. A Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway enrichment analysis of common targets was performed using R language software.

Statistical analyses
Statistical analyses were performed using the Prism software (GraphPad Software Inc., La Jolla, CA, USA). T-tests and one-way ANOVA were used for two-group and multiple group comparisons, respectively. The results are presented as mean ± standard deviation (SD) and P < 0.05 was considered statistically significant.

Icariin promoted osteogenic differentiation of MC3T3-E1 cells
To determine the effects of icariin on the proliferation of MC3T3-E1 cells, cell viability was measured in basal medium with different concentrations of icariin (0, 0.01, 0.1 and 1 μM) for 24, 48 and 72 h. As shown in Fig. 1A, MC3T3-E1 cell viability did not change significantly after icariin treatment.
ALP enzyme activity, Alizarin red S staining and qPCR were used to evaluate the effects of icariin on osteogenic differentiation of MC3T3-E1 cells by using different concentrations of icariin in osteogenic differentiation culture medium respectively. First, ALP enzyme activity observably increased after 7-days 1 μM icariin treatment (Fig. 1B).
Moreover, the results of Alizarin red S staining after 21-days icariin treatment indicated the increase of calcium deposition, especially at an icariin concentration of 1 μM. This result was confirmed by measuring absorbance at 562 nm after dissolving the calcium deposits ( Fig. 1C and  D). Furthermore, the expression of osteoblast markers in MC3T3-E1 cells was measured after 7 days of icariin-intervened osteogenic induction. The qPCR results showed that osteoblast markers (Alp, Bgp and Runx2) were significantly upregulated in MC3T3-E1 cells after icariin treatment compared with those in untreated cells (Fig. 1E-G). The qPCR results were consistent with the results of the Alizarin red S staining experiments. These results demonstrate that 1 μM icariin significantly promoted osteogenic differentiation of MC3T3-E1 cells. Thus, 1 μM icariin was adopted in the following experiments.

Eno1 was upregulated during icariin-induced osteogenic differentiation
Dysregulation of glucose metabolism is closely related to diabetic osteoporosis, and anti-osteoporotic medications, such as icariin, affect the incidence of diabetes mellitus and glucose metabolism [20,21]. To study the mechanism by which icariin affects osteogenic differentiation due to glycolytic disorders, we screened for potential targets in postmenopausal osteoporosis (PMOP) and diabetes mellitus (DM) using bioinformatics analysis. Of the 839 PMOP-related genes and 4033 DM-related genes identified from the target database, 693 potential genes were screened out at the intersection ( Fig. 2A). The KEGG analysis was further performed on these 693 target genes. Among all the enriched KEGG pathways, the hypoxia inducible factor (HIF)-1 pathway caught our attention due to its vital regulatory role in glucose metabolism (Fig. 2B) [22]. Moreover, the protein-protein interaction network was generated (Fig. 2C). Considering that Eno1 and GAPDH are downstream genes of the HIF-1 signalling pathway and that these two genes are enzymes on the glycolysis pathway, the two glycolytic targets were identified as promising targets regulating bone metabolism. Combined with our previous data showing altered expression of Eno1 in icariin-treated ovariectomised rats (data unpublished), we speculated that Eno1 may be a potential target for icariin regulation of osteogenic differentiation.
To verify our assumption, Eno1 protein and mRNA levels were measured. Compared with MC3T3-E1 cells cultured in blank induction medium, Eno1 mRNA level in MC3T3-E1 cells treated with osteogenic differentiation culture medium containing icariin went up (Fig. 2D). The expression of Eno1 was also upregulated after the addition of icariin, which was consistent with the qPCR results ( Fig. 2E-F).

Inhibition of Eno1 reduces icariin-induced osteoblast differentiation
To determine if icariin regulates osteoblast differentiation through Eno1, the Eno1 specific inhibitor, ENOblock, was used. To determine the effects of ENOblock on cell viability, CCK8 assays were performed at different concentrations of ENOblock (0.05, 0.5 and 5 μM) for 24, 48 and 72 h. As we can see, ENOblock did not change MC3T3-E1 cell viability (Fig. 3A).
Cells were further separated into blank osteogenic differentiation culture medium group, osteogenic differentiation culture medium containing ENOblock group, osteogenic differentiation culture medium containing icariin group and osteogenic differentiation culture medium containing both icariin and ENOblock group. Alizarin red S staining and qPCR were used to detect the differentiation of MC3T3-E1 cells in each group. As shown in Fig. 3B, icariin increased calcium deposition in MC3T3-E1 while ENOblock decreased calcium deposition. Icariininduced increase in calcium deposition was attenuated by ENOblock. These results were also confirmed by measuring the absorbance at 562 after dissolving the calcium deposits (Fig. 3C). Osteoblast markers (Alp, Bgp and Runx2) were significantly upregulated in MC3T3-E1 cells treated with icariin, while osteoblast markers were markedly decreased after treatment with ENOblock. The increased osteoblast markers induced by icariin were hindered to some extent by the administration of ENOblock (Fig. 3D-F). Collectively, we reckoned that Eno1 could be a target for icariin efficacy in osteogenesis.

Eno1 regulates the osteogenic differentiation of MC3T3-E1 cells through the BMP/Smad4 signalling pathway
To further investigate the way in which Eno1 regulated osteogenic differentiation, we examined the BMP/Smad4 signalling pathway, a widely accepted osteogenic pathway [23]. Icariin increased the protein levels of BMP2, BMP4, Smad4 and p-Smad1/5/9 compared with those of levels in the control group, while ENOblock decreased BMP2, BMP4, Smad4 and p-Smad1/5/9. In the icariin + ENOblock group, the levels of BMP2, BMP4, Smad4 and p-Smad1/5/9 were downregulated compared with those in the icariin-treated group (Fig. 4A-E). These results indicated that Eno1 took part in osteogenic differentiation via BMP/Smad4 signalling pathway.
Aerobic glycolysis is the main pathway for glucose metabolism in osteoblasts and provides energy for osteogenic differentiation, even in the presence of oxygen, akin to the Warburg effect in cancer cells [25]. Eno1, a glycolytic enzyme, is frequently upregulated in cancer cells and increases the Warburg effect [26]. Thus, Eno1 is a promising target for treating cancer [27]. Similar to cancer, inhibition of Eno1 leads to markedly depressed osteogenic differentiation, suggesting the potential role of Enol in regulating osteogenic differentiation. Furthermore, Eno1 is modulated by HIF-1α in many cancer cells [28][29][30]. As a glycolytic regulator, HIF-1α were testified to promote bone formation [31]. Our findings that Eno1 can regulate osteogenic differentiation are consistent with previous reports. Collectively, Eno1 may be an important factor in osteogenic differentiation.
Icariin was previously explored to augment osteogenesis in several ways, including promoting migration and regulating osteogenesisrelated signalling pathways and transcriptional factors [24,32,33]. In the present study, the promoted osteogenesis induced by osteogenic differentiation culture medium with icariin were significantly reduced by the addition of ENOblock, indicating that Eno1 plays an important role in icariin-regulated osteogenesis. Moreover, KEGG analysis showed that the HIF-1α signalling pathway is a potential target for icariin. Previous studies confirmed that icariin facilitates chondrocyte vitality by promoting HIF-1α expression and anaerobic glycolysis [34]. Thus, combined with our results, we assume that icariin augments aerobic glycolysis via upregulation of Eno1 to promote osteogenic differentiation. However, whether icariin-induced increased expression of Eno1 affects the HIF-1α signalling pathway still needs further exploration. BMP/Smad4 signalling is a key pathway leading to bone formation [23,35]. Icariin modulates osteogenic differentiation through the BMP/Smad4 signalling pathway [17,36]. Our data demonstrated that 1 μM icariin increased the expression of BMP2, BMP4, Smad4 and p-Smad1/5/9, in accordance with previous reports. We also demonstrated that BMP2, BMP4, Smad4 and p-Smad1/5/9 expression levels decreased in the ENOblock and icariin + ENOblock groups, suggesting that Eno1 might regulate osteogenic differentiation through the BMP/Smad4 signalling pathway. However, the detailed connection between Eno1 and the BMP/Smad4 signalling pathway has not been fully established.

Author contributions
Dingbang Xie and Hui Yan performed the experiments and drafted the manuscript. Yunteng Xu, Wanping Cai, Junkuan Zhuo, Zaishi Zhu, Haifeng Zhang, Yimin Zhang, Xin Lan helped to analyse the data and edit the manuscript.

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The ratio of p-Smad1/5/9 and Smad1/5/9 was displayed. All the data are shown as mean ± SD (n = 3) in three independent experiments. *P＜0.05, **P ＜0.01versus the blank control group, && P＜0.01, &&& P＜0.001 versus the icariin group.